Wound healing formulations containing human plasma fibronectin

ABSTRACT

Topical gel and cream formulations containing human plasma fibronectin are used for the healing of cutaneous wounds. The formulations provide slow release and increased contact time of fibronectin to the wound site leading to effective absorption of an effective wound healing amount of fibronectin in the skin.

FIELD OF THE INVENTION

The present invention relates to topical dosage forms containing humanplasma fibronectin for use in promoting wound healing in humans. Inparticular, the invention relates to the healing of chronic venousulcers.

BACKGROUND OF THE INVENTION

Fibronectin is a large glycoprotein containing around 5% carbohydrate.The characteristic form of plasma fibronectin is a disulfide-bondeddimer of 440,000 daltons, each subunit having a molecular weight ofabout 220,000 daltons. Normally found in plasma at a concentration ofabout 300 μg/mL, fibronectin is extracted and purified using a methoddescribed by Hynes¹. Plasma fibronectin is also known by various othernames, including cold-insoluble globulin, anti-gelatin factor, cellattachment protein, cell spreading factor, and opsonic α2-surfacebinding glycoprotein. These names reflect biological activities offibronectin such as cell recruitment, opsonization of particulatedebris, and promotion of wound contraction. Reviews on structure andactivities of fibronectin have been published elsewhere²,3.

Wound healing is usually divided into three phases: the inflammatoryphase, the proliferative phase, and the remodeling phase. Fibronectinhas been reported to be involved in each stage of the wound healingprocess, particularly by creating a scaffold to which the invading cellscan adhere. Initially, many mediators, such as fibronectin andfibrinogen, are released to the wound site. Fibronectin promotesinflammatory cells migration into the wound and debris phagocytosis bythe monocytes. Thereafter, angiogenesis and reepithelialization takeplace. At this stage fibronectin exerts chemotactic activity onendothelial cells, and promotes the migration of epithelial cells andfibroblasts onto the basal membrane. Fibronectin also appears to be anessential component of the remodeling phase where it plays a major rolein the organization of collagen fibrils. The fibrillar collagenultimately forms fibrous bundles that greatly enhance the tissue tensilestrength, leading to wound closure.

Topically applied plasma fibronectin has been reported as being usefulfor increasing the rate of wound healing such as in corneal wounds⁴,5and leg ulcers⁶. However, no one has described a suitable topicalcarrier for use in treating wounds that can ensure the delivery of aneffective amount of fibronectin. A major limiting factor in developingan effective topical dosage form of a drug is not only having an activedrug, but also having a formulation that allows the passage of theactive drug from the carrier (cream, ointment, gel, etc.) into the siteof delivery (which in the case of the present invention is a skinwound). Very active drugs, such as growth factors, may have notherapeutic value if the topical formulation does not allow the drug tomove from the semi-solid carrier into the wound. Therefore, it would behighly desirable to develop a formulation which would maximize thecontact time of the fibronectin with the wound and also control therelease of fibronectin to the wound, thereby leading to high absorptionvalues. The present invention provides such delivery system in the formof aqueous gels and a cream.

SUMMARY OF THE INVENTION

The present invention provides aqueous gel formulations and one creamformulation containing fibronectin and their use for the delivery of aneffective wound healing amount of fibronectin to a wound site. The gelformulation comprises a water soluble, pharmaceutically acceptablepolymer which is prepared from an effective amount of fibronectin.Examples of such compounds include: vinyl polymers, e.g. polyacrylicacid; polyoxyethylene-polyoxypropylene block copolymers, e.g. poloxamer;and cellulose derivatives, e.g. hydroxypropylcellulose (HPC). Thepolymer provides viscosity values between 50,000 and 1,000,000 cps atroom temperature. The cream formulation is prepared from a commerciallyavailable cream base i.e., Schering® base (Schering Canada Inc.,Point-Claire, Quebec), which has viscosity values between 60,000 to80,000 cps at room temperature.

Many advantages are attributed to these dosage forms. Gel and creamformulations of the present invention release effective amounts of awound healing promoter. Other advantages of gel formulations include:ability to keep the wound moist (which results from the high watercontent of the gels), ease of application and removal (by washing) fromthe wound. They also provide a cool feeling when topically applied whichcan increase patient comfort.

The slow release system of gel formulations of the present inventionprovides extended release of fibronectin to the wound site. Thisproperty of these formulations permits less frequent application to thewound resulting in less disturbance to the healing process. Suchformulations maintain fibronectin delivery for up to 24 hours; butaccording to kinetic data obtained from permeation studies, a "twice aday" therapeutic schedule is a preferred embodiment of the presentinvention.

Formulation of topical dosage forms intended for the incorporation offibronectin should respect several quality criteria. All components ofthe preparation including solvent, gelling agent and preservative shouldbe nontoxic for the wound and compatible with the drug. The finalproduct should promote optimal release of the drug to its site ofaction, be of adequate consistency to enhance contact time of the drugwith the wound and be sterile.

The preferred formulations of this invention can be used with otherwound healing promoters having a composition similar to fibronectin,such as proteins of similar size (thrombospondin, laminin, vitronectin,fibrinogen) or smaller size (such as peptides including growth factors).

The preferred formulations can be correlated with the results ofevaluating the formulations using an in vitro diffusion cells systemconsisting of a rigid receptor containing a deepithelialized skinsample, the deepithelialized side facing upwards into a donorcompartment and the dermal side facing downwards into a receptorcompartment. The receptor compartment is connected to a circulatingbuffer circuit, with the buffer temperature maintained at 37° C. whilethe skin surface is at about 32° C. Preferred compositions will have anAbs value of greater than 7.8, preferably at least about 13.40.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the cumulative absorption of radio-labelled fibronectinover time from various gel formulations containing Carbopol® P-934carbomer (0.375%), Pluronic® F-127 poloxamer (20.0%), sodiumcarboxymethylcellulose (CMC 3.0%), and from the control (phosphatebuffered saline solution).

FIG. 2 depicts cutaneous absorption of radio-labelled fibronectin fromvarious dosage forms and from the control (phosphate buffered salinesolution) at time=12 hours.

FIG. 3 depicts the electrophoresis of human plasma (FN) incorporated ina Carbopol® carbomer gel (Carbopol® P-934 carbomer 0.375% +chlorocresol0.1%) after 0, 2, 6, and 8 months. Section A: Recovery of FN after agelatin-binding test. Section B: Integrity of FN after 240 days ofstorage in gel at 4° C. It should be noted that in section B, theresolution of the band is affected by the presence of contaminants suchas Carbopol® carbomer in the specimen.

FIG. 4 shows a plot of dermal absorption versus viscosity from differenttopical preparations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides dosage forms that are speciallyformulated for the therapeutic use of fibronectin as a topical woundhealing promoter. The dosage forms selected for topical applicationsshould ideally release large amounts of fibronectin, be sterile andnon-toxic for the wound. Several factors such as physico-chemicalproperties of the glycoprotein as well as clinical utilization criteriamust be considered when compounding these formulations.

Among these limitations, the major one relates to the solubility offibronectin in water which is poor and therefore mitigates against thepreparation of concentrated gels or creams. Fibronectin is only slightlysoluble in water and may precipitate at concentrations as low as 5 mg/mLin aqueous solution. Its solubility is also affected by pH changes andlow temperatures. In the same way, formulations that require thedispersion of polymer powder in the fibronectin solution under agitationcannot be prepared easily since precipitation of the glycoprotein mayoccur. Under agitation, fibronectin may aggregate and form long mats ofinsoluble material. Viscosity must be optimal in order to permit asufficient adherence to the wound as well as good release capabilities.

Temperatures over 60° C., which are frequently required to providesterile preparations, denature fibronectin. Since a terminalsterilization process cannot be performed on the final product, thepreparation of concentrated bases of vehicles without fibronectin isusually unavoidable. Portions of these sterile bases are then dilutedwith a definite amount of a solution of fibronectin. To achieve adequatedispersion of fibronectin into semi-solid dosage forms, an incorporationstep involving agitation is often required which can lead to theprecipitation of the drug.

Gelling agents such as Carbopol® carbomer and poloxamer can circumventthis problem since they are sterilized before gelation under aliquid-like, viscous form. A highly concentrated preparation ofCarbopol® carbomer is prepared and autoclaved. As described below, thesolution of fibronectin which also contains the polymerization promoter(NaOH) is then mixed in syringes with the base of Carbopol® carbomer,building-up the gel during the dispersion of the drug into it. In thecase of poloxamer, the polymer is added to the drug solution and allowedto dissolve at 4° C., a temperature at which it maintains its fluid-likeaspect. Sterilization of this solution from bacteria is performed at 4°C. using a 0.22 μm filter.

A non-toxic, non-sensitizing preservative compatible with formulationcomponents is added to the dosage form in a preferred embodiment of theinvention. All of the above conditions are respected in the preferreddosage forms described in detail as follows.

An effective wound healing amount of human plasma fibronectin for use inthe present invention is within the range of 0.05 to 0.5% by weight andmore preferably between 0.2 and 0.4%. Fibronectin is isolated from humanplasma by using a gelatin-Sepharose affinity chromatography procedure.In this method, gelatin is covalently coupled to Sepharose 4B after CNBractivation. The binding capacity for human plasma fibronectin providedby this system is >1 mg/ml of gel.

Autologous, homologous human plasma fibronectin or fibronectin obtainedfrom recombinant-DNA technology may be used in the present invention¹,7.Should homologous plasma fibronectin be used, lots prepared fromdifferent donors would have to be tested for atypical antibodies,hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus(HIV), human T-cell lymphotrophic virus (HTLV), cytomegalovirus (CMV)and syphilis. These tests have to be performed on donors just prior tothe donation and 6 months thereafter. In the meantime, donor plasma mustbe kept frozen at -20° C. Furthermore, special steps should be taken inorder to inactivate potential viruses. An inactivation method using tri(n-butyl) phosphate/Tween-80 or tri (n-butyl) phosphate/Triton X-100(solvent/detergent method) should be performed on all plasmadonations⁷,8.

In the gel formulation for topical wound healing, the viscosity may bewithin the range of 50,000 to 1,000,000 cps, more preferably between100,000 and 650,000 cps. In the cream formulation, the viscosity may bewithin the range of 60,000 to 80,000 cps. All viscosity values are incentipoises (cps) as measured using a Brookfield viscometer. Assays wereperformed at 0.5 rpm and at room temperature.

In one embodiment of the present invention, the gel formulation maycomprise 0.25 to 1.0% by weight polyacrylic acid having a molecularweight of about 740,000 to 5,000,000. In a preferred embodiment, thepolyacrylic acid is present at 0.35 to 0.75% by weight and has aviscosity of about 350,000 cps. The pH of the polyacrylic acid gelshould be within the range of 5 to 8 and more preferably between 6.5 and7.5. Polyacrylic acid polymer, also known as carbomer, is sold under thetrademark Carbopol®. The preferred grade of Carpobol® carbomer is P-934.

In another embodiment, the gel formulation may comprise 18 to 35% byweight polyoxyethylene-polyoxypropylene block copolymer having amolecular weight of about 2,000 to 13,000. In a preferred embodiment,the polyoxyethylene-polyoxypropylene block copolymer is present at 18 to25% by weight and has a viscosity of about 450,000 cps at roomtemperature. The pH of the block copolymer gel should be within therange of 6 to 8 and more preferably between 6.5 and 7.5.Polyoxyethylene-polyoxypropylene block copolymer, also known aspoloxamer, is sold under the trademark Pluronic®. The preferred gradePluronic® poloxamer is F-127 (poloxamer 407).

In a further embodiment, the gel formulation may comprise 1 to 5%cellulose derivative which may be hydroxypropylcellulose (HPC) and has aviscosity of about 25,000 to 150,000 cps. HPC has a molecular weight ofabout 370,000 to 1,150,000. In a preferred embodiment, the cellulosederivative is present at 2.0 to 4.0% by weight and has a viscosity ofabout 150,000 cps for HPC. Cellulose derivatives used in the presentinvention are commonly known as Klucel for HPC. Preferred grade isKlucel-HF.

In a further embodiment, a cream formulation is prepared from acommercially available cream base i.e., Schering® base. This cream base(oil in water emulsion) contains ceteth-20, cetostearyl alcohol,chlorocresol, mineral oil, monobasic sodium phosphate, phosphoric acid,sodium hydroxide, water and white petrolatum. The viscosity of thepreparation can be modified by varying the content of water andpolyethylene glycol.

Formulations of the present invention contain an aqueous phase incombination with a protein and thus are prone to attack by bacteria andfungi. Microbial growth not only spoils the formulation but is apotential toxicity hazard and a source of infection for patients. Eventhough microbial growth is less likely to be dangerous when it occurs ina topical preparation, it is especially important to preserve topicalswhich patients have to apply to broken or inflamed skin. Viscositydegradation reported with some polymers when exposed to microbialcontamination is also of concern. So, a preservative should be added tothe preparation to guarantee long term sterility and stability. Thepresent invention provides gels that comprise a preservative selectedfrom phenol or the para-hydroxybenzoate compounds. In one embodiment,the gel formulation may contain 0.1 to 0.2% by weight chlorocresol, aphenol derivative or 0.01 to 0.3% by weight p-hydroxybenzoate as methyl-and propylparaben. In another embodiment, the cream formulation contains0.1 to 0.2% by weight chlorocresol.

Stabilizers may be added to the formulation in order to provide stablecompositions of fibronectin. They may help to preserve biologicalactivities on a long term basis and may improve water solubility offibronectin. Among these agents, albumin, disaccharides such as sucrose,and cyclic oligosaccharides such as cyclodextrins are stabilizers ofchoice. These agents can be used either alone or in combination. Humanalbumin is preferable in terms of antigenicity and should be free frommicrobial contamination. Cyclodextrins of the β group (7 glucose units)are of choice and hydroxypropyl-β-cyclodextrin is preferable. Theformulation may comprise 0.01 to 0.1% by weight albumin, preferably 0.01to 0.05%; and/or 0.5 to 5.0% by weight sucrose, preferably 3.0 to 5.0%;and/or 1.0 to 10% by weight hydroxypropyl-β-cyclodextrin, preferably 2.0to 5.0%.

Some authors have suggested that protease activity in some chronicwounds may cause degradation of adhesion proteins such as fibronectinand prevent cell adhesion necessary for normal wound closure⁹.Metalloproteases and serine proteases have been identified in chronicwound fluid⁹,10 and fibronectin has been reported to be highly sensitiveto cleavage by proteases¹¹. Protection of the integrity of fibronectinmay be accomplished by the addition of protease inhibitors in the dosageform. The present invention also provides formulations that may comprisea metalloprotease inhibitor such as EDTA and/or a serine proteaseinhibitor such as aprotinin (Trasylol®, Miles) with this aim in view. Inone embodiment, the dosage form may comprise 0.01 to 1.0% by weight EDTAand/or 1.5 to 45.0 Inh U % by weight aprotinin where 1 Inh U=26Kallikrein inhibitor units.

Formulations of the present invention can be applied to the wound siteby any suitable means which assures that the wound surface will beentirely covered. For example, it can be directly applied to the woundsite or used to coat fibers of an absorbent gauze dressing to form awound healing bandage which may then be placed on a wound.

Examples which follow are intended to illustrate further aspects of theinvention and are not to be construed as limiting its scope in any way.

EXAMPLE 1 Isolation of Fibronectin from Human Plasma

1) A sterilization step is mandatory for all homologous plasmadonations. In order to inactivate potential viruses, a sterilizationprocedure using the s solvent/detergent method is used. 1% tri (n-butyl)phosphate (TNBP) and 1% Triton X-100 are added to the plasma for 6 hoursat 24° C. After that, soybean oil is added to the plasma and allowed tobe mixed for at least 30 minutes in order to extract TNBP. ResidualTriton will be eliminated by dialysis.

This first step is skipped if autologous plasma is used.

2) A gelatin-Sepharose 4B column is first prewashed with a Tris-HClsolution in order to equilibrate the gel.

3) The plasma is diluted (1:1) with a Tris-HCl solution and pumpedthrough the column in the presence of phenylmethylsulfonyl fluoride0.001M (PMSF) for about 15 hours at 4° C.

4) The column is then washed three times in order to elutenonspecifically bound plasma proteins from the gel. All washing stepsare performed using a Tris-HCl pH 7.5 solution. A 1M NaCl solution isadded to the second washing step to elute contaminants.

5) Elution of fibronectin is carried out by using 0.1M Na acetate+1M KBrsolution.

6) Two dialysis steps are then performed to eliminate contaminants(Triton X-100, KBr, Na acetate). Dialysis versus PBS and sterile waterare respectively done.

7) Solution is concentrated by ultrafiltration under nitrogen pressure.

8) Terminal sterilizing filtration using a 0.22 μm filter is done towarrant sterility.

9) Fractions are aliquoted and frozen at -20° C. until theirincorporation into the topical dosage form.

EXAMPLE 2 Polyacrylic Acid Gels

Polyacrylic acid (carbomer) gels (Carbopol®, carbomer, B F Goodrich)were prepared. Carbomer is a polymer derived from acrylic acid. It is ahigh molecular weight polymer (740,00 to 5,000,000) that gelifies whenneutralized by strong alkalis (NaOH) or amines (triethanolamine). Itforms gels at relatively low concentrations, that is as low as 0.25%,and its viscosity is strongly reduced by the addition of electrolytes.

Preferred grade of polyacrylic acid is Carbopol® 934-P carbomer atconcentrations ranging from 0.35 to 0.75% (w/w). Lower concentrationsare insufficient to promote adherence to the wound and higherconcentrations reduce the release of fibronectin from the gel. Viscosityof polyacrylic acid gels is stable between pH 6 to 8 with a preferred pHrange between 6.5 to 7.5. Viscosity is reduced in the presence of strongelectrolytes.

A polyacrylic acid gel containing (w/w) fibronectin 0.2%, Carbopol®934-P carbomer 0.375%, and chlorocresol 0.1% was prepared as follows:chlorocresol (1.0 g) was dissolved in warm (65° C.) deionized water (95mL) under slow agitation. When the chlorocresol is completely dissolved,the solution is cooled at room temperature while maintaining agitation.Carbopol® 934-P carbomer (3.75 g) was then added, dispersing it slowlyon the surface of the solution, and mixed with a paddle type stirrer forabout 3 hours. This dispersion was then autoclaved to provide a sterileconcentrated gel base (3.75% w/w). A stock solution of fibronectin 2.2mg/mL (90 mL) was filtered through a 0.22 μm acetate filter. Apolymerization promoter, sodium hydroxide, was added to the fibronectinsolution in an amount that will neutralize a 10 g portion of theCarbopol® carbomer 3.75% dispersion, that is 1250 μL of NaOH 3M. Thestock solution of fibronectin and Carbopol® carbomer dispersion weremixed into syringes taking care to avoid introducing air bubbles andcontamination in an aseptic environment, such as under a laminar flowhood. This preparation provides a clear, preserved gel (100 g) offibronectin free from microorganisms with viscosity of about 350,000cps.

This gel formulation was applied twice a day on leg ulcers in a pilotstudy in humans and showed an enhanced rate of wound healing without anyadverse effect.

EXAMPLE 3 Polyoxyethylene-polyoxypropylene Block Copolymer Gels

Polyoxyethylene-polyoxypropylene block copolymer (poloxamer) gels(Pluronic® poloxamer, BASF Wyandotte) were prepared. Preferred grade ofpoloxamer is Pluronic® F-127 poloxamer at concentrations ranging from 18to 25% (w/w). Poloxamer® F-127 poloxamer is a low molecular weightpolymer (2,000 to 13,000) which exhibits thermal gelationcharacteristics. Gelation occurs when the concentration reaches 18%poloxamer. The viscosity of poloxamer is proportional to theconcentration of the polymer, type of polymer used (molecular weight)and temperature. Fluid at 4° C., the polymer gelifies with increasingtemperatures, providing high viscosity values at room temperature. Incontrast to Carbopol® carbomer, the addition of ions enhances theviscosity of the preparation.

Concentrated aqueous solution (20 to 30%) have been reported to show adramatic increase in viscosity when heated from 4° C. to bodytemperature. Furthermore, if the ionic strength of the solution isincreased, the viscosity is increased more rapidly with risingtemperature. Several grades are available but the F-127 grade is theleast toxic and gelation can occur at lower concentrations. Gels ofpoloxamer prepared in this invention are low viscosity solutions at 4°C. and gelify rapidly when they are warmed to body temperature.

A poloxamer gel containing (w/w) fibronectin 0.2% and Pluronic® F-127poloxamer 20% was prepared as follows: a stock solution of fibronectin2.2 mg/mL (80 mL) was filtered through a 0.22 μm acetate filter.Pluronic® F-127 poloxamer (20 g) was added to 80 mL of the fibronectinsolution and allowed to dissolve without agitation at 4° C. for about 3days. The resulting solution (100 g) is very liquid-like. Gelationoccurs instantly when the solution comes into contact with the wound. Asterilizing filtration process performed at 4° C. could also be appliedto the final solution if sterile poloxamer powder cannot be obtained.Viscosity varies from not detectable values at 4° C. to 450,000 cps atroom temperature.

EXAMPLE 4 Cellulose Derivative Gels

Hydroxypropylcellulose (HPC) gels were prepared. In order to illustratethis type of formulations, the preparation of a HPC 3% gel is describedas below. Preferred grade is Klucel-HF hydroxypropylcellulose atconcentrations ranging from 2 to 4% (w/w).

A gel formulation containing (w/w) fibronectin 0.1%, HPC 3% and parabenswas prepared as follows: methylparaben (0.05 g) and propylparaben (0.02g) were dissolved in warm deionized water (94 mL). HPC powder wassterilized by using a dry heat sterilization process. HPC (6 g) was thendispersed in this solution and allowed to be mixed with a paddle typestirrer for about 3 hours. This provides a sterile concentrated gel base(6% w/w). A stock solution of fibronectin 2 mg/mL (50 mL) was filteredthrough a 0.22 μm acetate filter. Fibronectin solution (50 mL) was thenslowly added to a portion (50 g) of this concentrated base using thelow-speed shaft of the stirrer. This provides a preserved gel (100 g)with viscosity of about 150,000 cps.

EXAMPLE 5 Cream Formulation

A cream formulation containing (w/w) fibronectin 0.1%, sterile creambase (Schering® base, Schering) and chlorocresol 0.1% was prepared asfollows: a stock solution of fibronectin 2 mg/mL (50 mL) was filteredthrough a 0.22 μm acetate filter. Fibronectin solution (50 mL) was thenadded slowly to a portion (50 g) of the cream base using the low-speedshaft of a stirrer. This provides a preserved cream (100 g) withviscosity of about 70,000 cps.

EXAMPLE 6 Kinetics of Release from Different Topical Dosage Forms

The effectiveness of each topical formulation to release fibronectin wasevaluated using an in vitro diffusion cell system. Permeation studieswere all performed on human breast and abdominal deepithelialized skinsamples obtained from breast reduction and abdominal lipectomysurgeries. A 8 μm section was removed from the epidermal surface of theskin using a dermatome (1/10,000 scissor scale) and the dermal side wascarefully cleaned of any adhering subcutaneous tissues and/or bloodvessels. Deepithelialized human skin was used in order to reproduce thepathological condition met in chronic venous ulcers where the epidermislayer is absent.

The diffusion cell system selected consisted of a rigid receptorcontaining the skin sample, the deepithelialized side facing upwardsinto the donor compartment and the dermal side facing downwards into thereceptor compartment. The receptor compartment was connected to acirculating buffer circuit. The buffer temperature was maintained at 37°C. while the skin surface was at about 32° C. Each analysis wasperformed on a 0.64 cm² skin sample using a 100 μL aliquot of ¹²⁵-fibronectin topical formulation specimen. After the experiment, theskin was removed from the diffusion cell, washed 10 times with a 8 mLwater volume by wash, and analyzed for its content of radioactivity in agamma radioactivity counter. The total amount absorbed (dermis+receptorcompartment) divided by the dose applied gave the percent absorption.

All dosage forms were made in salt-free solution since viscosity valuescould have been influenced by the presence of electrolytes. Forinstance, viscosity values of carbomer gels are reduced in the presenceof strong electrolytes in contrast with poloxamer gels which are moreviscous when electrolytes are added to the preparation.

Several authors have compared percutaneous absorption studies using invitro and in vivo techniques to establish the reliability of resultsusing these methods²,3,4. These comparisons have clearly shown that invitro studies can accurately reflect the living state. Statisticanalysis applied to our experiments has demonstrated a good correlationvalue between studies performed on skin obtained from different sources.These data have shown that the origin of the skin did not have anyeffect on results.

Percutaneous absorption studies are usually performed on intact skin andare designed to evaluate the release of a substance from a topicalvehicle and its absorption through the major cutaneous barrier, that isthe stratum corneum. In cutaneous ulcers, the barrier effect of thestratum corneum is absent. With this pathological condition, only thediffusion from the dermatological vehicle will be a major determinantfor the ulterior penetration of the drug into the dermis. The diffusioncell system described above is a suitable in vitro model for cutaneousulcers.

Kinetic data of the release of fibronectin from various dosage formswere obtained at 4, 12, and 24 hours. Table 1 summarizes these data fort=12 hours. The control consisted of ¹²⁵ I-fibronectin in phosphatebuffered saline solution, pH 7.4. Liposomes used in the Carbopol® 934-Pcarbomer (1%)+liposomes (15%) formulation (Lipogel) were made fromProliposomes lipsomes (Pro-lipo 3090 SH⁰, Lucas Mayer, France).Cellulose derivatives are identified as CMC for sodiumcarboxymethylcellulose and HPC for hydroxypropylcellulose. Dermabase®base (Borden, Ltee., Don Mills, Ontario, Canada) and Schering® base arecream bases available on the market and were diluted 1:1 for theseexperiments. The symbol [] refers to components concentration and "Absvalue" to the percentage of radiolabelled fibronectin found in thedermis after an exposition time of 12 hours.

                  TABLE 1                                                         ______________________________________                                        Formulation       [ ]          Abs value                                      ______________________________________                                        Control                        24.75%                                         Lipogel                        3.70%                                          Dermabase         (1:1)        5.80%                                          CMC               3%           6.70%                                          Carbopol 934 P + glycerol                                                                       0.375%/10%   7.80%                                          (Carbogly)                                                                    Schering base     (1:1)        9.90%                                          Pluronic F-127    20%          12.80%                                         Carbopol 934 P    0.375%       13.40%                                         HPC               3%           15.20%                                         ______________________________________                                    

FIG. 1 plots kinetic data of three gel dosage forms and control solutionover time. From this graph it can be seen that the absorption processtend to be more important between time 0 and 12 hours than between time12 and 24 hours, suggesting that two applications per day could releasemore fibronectin than an once a day schedule.

FIG. 2 depicts cutaneous absorption of radiolabelled fibronectin fromvarious dosage forms and from control at time=12 hours. The Dunnettstatistic test was used to identify statistically significantdifferences between Carbopol® carbomer gel and other formulations. Thistest has also shown significant differences between Lipogel, Carbogly,and Carbopol® carbomer gel formulations, results that can be correlatedwith those of effectiveness obtained during clinical trials (see example8). The efficacy of the Carbopol® carbomer gel formulation isparticularly surprising since Carbopol® carbomer gel has a higher degreeof viscosity than many of the other formulations studied. Alsonoteworthy are the difference in Abs value between the Carbopol®carbomer gel and CMC formulations since they both share the same degreeof viscosity.

FIG. 4 show that a clear relationship between viscosity and absorptiondoes not always exist, when considering the some of the preparations forwhich viscosity values were determined. For instance, Dermabase® basewhich has a relatively low viscosity (119,000 cps) when compared toCarbopol® carbomer gel (411,300 cps) presents poor release capabilities(5.80%) when compared to Carbopol® carbomer gel (13.40%).

EXAMPLE 7 Stability of Fibronectin in Gel

Biological activity and integrity of the macrostructure of fibronectinin gel formulations were evaluated (FIG. 3). Assays were performed on aspecimen of gel containing (w/w) fibronectin 0.2%, Carbopol® P-934carbomer 0.375%, and chlorocresol 0.1%. The specimen had been kept at 4°C. for 32 weeks.

Electrophoresis techniques were used in order to determine the integrityof macrostructure of fibronectin in gel. After the specimen of gel wasdissolved in 1M NaCl+Tris-HCl pH 7.4 solution, it was allowed to migrateon an 7.5% acrylamide gel according to the method of Laemmli("Denaturing (SDS) discontinuous gel electrophoresis: Laemmli gelmethod," pages: 10.2.4-10.2.9, Current Protocols in Molecular Biology1994). Compared with a fresh standard solution (column 0), resultsshowed that close to 100% of the fibronectin can be identified aroundthe 220,000 (column B) band indicating that very little, if any,degradation occurs.

Biological activity was evaluated using an affinity chromatography test.Gelatin-binding is one of these biological activities that can beassessed with relative ease. After a specimen of gel was dissolved in a1M NaCl solution, a known amount of this viscous solution was placed inan Eppendorf tube in the presence of gelatin- Sepharose 4B and thenvortexed. The content was further rinsed with a fresh 1M NaCl solution,centrifuged and the supernatant discarded in order to eliminatecontaminants such as Carbopol® carbomer and chlorocresol that came fromthe dissolution of the gel. Fibronectin was eluted from thegelatin-sepharose 4B using a 1M KBr solution. The fraction collected wasallowed to migrate on an 7.5% acrylamide gel according to the method ofLaemmli. The band was then evaluated with respect to its content offibronectin using a densitometric scanning assay. The specimen collectedcould also be evaluated spectrophotometrically using optical density atwave length λ=280 nm.

Compared with a freshly prepared gel of fibronectin (column 0), it canbe seen that a large amount (80%) of fibronectin was recovered from thespecimen of gel formulation (column 8 months) indicating thatgelatin-binding activity of the glycoprotein can be preserved for a longperiod of time in this dosage form.

EXAMPLE 8 Clinical Trials: Treatment of Chronic Leg Ulcers

We have conducted four clinical trials (pilot studies) to investigatethe usefulness of different dosage forms containing exogenous humanplasma fibronectin in the treatment of chronic venous ulcers of lowerlimbs. In these trials autologous plasma fibronectin was used andpatients with ulcers that were resistant to the conventional therapy forat least three months were selected.

The specific objective of the first experiment was to determine theeffectiveness of topically applied fibronectin as a wound healingpromoter. Seven patients were included in this study and were instructedto "flood" the wound area with a solution of fibronectin 1 mg/mL (0.1%)in PBS (phosphate buffered saline) twice a day. After two months ofregular application of that solution, five of these patients presentedwith a dramatic decrease in their wound size, specifically at least 75%reduction of the integrated surface area.

A second experiment was designed to evaluate the effectiveness of asemi-solid dosage form which contained by weight fibronectin 0.1%,encapsulated in liposomes 15%, which in turn were incorporated inCarbopol® carbomer (1%) formulation known as Lipogel. Hypothesis wasthat if the contact time of the glycoprotein with the wound could beenhanced, a more rapid decrease in the healing time could theoreticallybe observed. Six patients were included in this study and they had toapply the formulation to their wound twice a day. None presented asubstantial decrease of their wound size during the following threemonths of regular treatment.

In an attempt to improve the dosage form, an experiment was undertakento evaluate the therapeutic potential of a topical gel formulationcontaining (w/w) fibronectin 0.2% incorporated in Carbopol® carbomer0.375% and glycerol 10% (Carbogly). Glycerol had been added to theformulation in order to take advantage of its humectant effect whichcould be beneficial to the wound. Eleven patients were recruited forthis study and they also had to apply the gel twice a day. Among thesepatients, 27% had a regression of more than 50% of their wound sizeafter three months of treatment.

Results from the permeation studies may explain, at least in part, whatcould have occurred in previous experiments. FIG. 2 shows thatpreparations such as Lipogel and Carbopol® carbomer+glycerol do not leadto high absorption values. In contrast, Carbopol® carbomer 0.375%without glycerol provides significantly higher absorption values(p<0.001). The solution used in the first experiment is identified asthe control in this graph. This last preparation provides the highestrelease capabilities but it does not represent a formulation that couldbe useful to patients owing to its fluid consistency.

Considering these results, a formulation containing fibronectin 0.2%(w/w) in Carbopol® carbomer 0.375%, without glycerol was investigated ineight patients. According to clinical and permeation studies, thisformulation is the preferred carrier using Carbopol® carbomer that isavailable for the use of fibronectin in topical wound healing.Preliminary data showed that 50% of patients studied presented aregression of more than 50% of their wound size within three months oftreatment, including two complete responses (100% healing) that occurredwithin the first eight weeks of treatment. The present invention alsoprovides other formulations that are as useful as this one using thepermeation study deacribed in Example 5 as a model system to test thevarious formulations.

EXAMPLE 9 Case Reports

To illustrate the efficacy of the formulation containing fibronectin andCarbopol® 934-P carbomer 0.375% (w/w), we present two specific cases ofchronic venous leg ulcer. These cases are of interest in that the firstcase was highly resistant to conventional therapy and the second casewas a large ulcer. Factors such as duration and surface area have beenidentified by several authors as playing a major role in the prognosisof the venous ulcer.

Case 1

A 37-year-old woman presented with a ten-year history of chronic venousulcer of the right lower limb. Her medical history was not significantexcept for four episodes of phlebitis. The last episode occurred duringpregnancy and ultimately resulted in an ulcer. Review of medicaltreatments that were tried revealed the use of topical antiseptics,elastic stockings, and skin grafting without any positive result.

The patient presented to our clinic with a 1.60 cm² aching wound.Despite the fact that her ulcer was relatively small, it appeared highlyresistant to therapy. Six weeks after starting the application of thegel of fibronectin, a 92% reduction of her wound size could be observed.Complete reepithelialization was noted after a ten-week course oftreatment. A follow-up visit scheduled one month later revealed nodeterioration in her wound condition.

Case 2

A 39-year-old man presented with a seven-month history of chronic venousulcer of the left leg. His medical history was not significant exceptfor a saphenectomy of the left lower limb twelve years before. Topicalantibiotics were prescribed to the patient without any effect on hiswound size.

He presented to our clinic with a 10.5 cm² ulcer resulting from a localtrauma. Lymphedema of the left lower limb was important and a largecrusty necrotic layer bordered the wound. The patient's occupationconstrained him to remain standing for long periods of time. Althoughthis situation probably worsened his wound condition, it could not beeliminated.

After four weeks of regular application of a placebo gel and normalsaline, the wound size increased to 21.5 cm² as a consequence of localdebridement. The placebo gel comprised 0.375% Carbopol® 934-P carbomer,0.1% chlorocresol, purified water and NaOH to adjust the pH. Activetreatment with the fibronectin-containing Carbopol® carbomer gelformulation was begun at this time. Maximum wound size was noted sixweeks later (37.5 cm²), revealing a larger ulcer than initially assumed.The wound healing process took place between six to eight weeks and wascompleted after 31 weeks of active treatment. A follow-up visitscheduled one month later revealed no deterioration in his woundcondition.

Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will become apparent to those skilled in the art. It istherefor understood that numerous variations of the invention can bemade which are well within the scope and spirit of this invention asdescribed in the appended claims.

REFERENCES

1--Hynes, R. O., Methods for identification of fibronectin (chap. 2,page 12), IN: Fibronectins New York: Springer-Verlag, 1990.

2--Hynes, R. O., Methods for identification of fibronectin (chap. 2,pages 7-23) and Wound healing, inflammation, and fibrosis (chap. 14,pages 349-64), IN: Fibronectins New York: Springer-Verlag, 1990.

3--Brotchie, H., Wakefield, D. Fibronectin: Structure, function andsignificance in wound healing. Australas J Dermatol 1990; 31:47-56.

4--Nishida, T., Nakagawa, S., Awata, T. et al., Rapid preparation ofpurified autologous fibronectin eyedrops from patient's plasma. Jpn JOphthalmol 1982;26:416-24.

5--Phan, T. M., Foster, C. S., Boruchoff, S. A. et al., Topicalfibronectin in the treatment of persistent corneal epithelial defectsand trophic ulcers. Am J Ophthalmol 1987;104: 494-501.

6--Wysocki, A., Baxter, C. R., Bergstresser, P. R. et al., Topicalfibronectin therapy for treatment of a patient with chronic stasisulcers. Arch Dermatol 1988; 124:175-7.

7--Edwards, C. A., Piet, M. P. J., Chin, S. et al., Tri (n-butyl)phosphate/detergent treatment of licensed therapeutic and experimentalblood derivatives. Vox Sang 1987; 52:53-9.

8--Horowitz, B., Bonomo, R., Prince, A. M. et al.Solvent/detergent-treated plasma: a virus-inactivated substitute forfresh frozen plasma. Blood 1992; 79: 826-31.

9--Grinnell, F., Ho, C. H., Wysocki, A., Degradation of fibronectin andvitronectin in chronic wound fluid: Analysis by cell blotting,immunoblotting, and cell adhesion assays. J Invest Dermatol 1992; 98:410-6.

10--Chen, W. Y. J., Rogers, A. A., Lydon, M. J., Characterization ofbiologic properties of wound fluid collected during early stages ofwound healing. J Invest Dermatol 1992; 99: 559-64.

11--Berman, M., Manseau, E., Law, M. et al., Ulceration is correlatedwith degradation of fibrin and fibronectin at the corneal surface.Invest Ophthalmol Vis Sci 1983; 24: 1358-66.

12--Franz, T. J., Percutaneous absorption. On the relvance of in vitrodata. J Invest Dermatol 1975: 64:190-95.

13--Bronaugh, R. L., Stewart, R. F., Congdon, E. R. et al. Methods forin vitro percutaneous absorption studies. I: Comparison with in vivoresults. Toxicol Appl Pharmacol 1982;62:474-80.

14--Bronaugh, R. L., Stewart, R. F., Methods for in vitro percutaneousabsorption studies IV: The flow-through diffusion cell. J Pharm Sci1985: 74:64-67.

I claim:
 1. An aqueous gel formulation for healing chronic wounds,comprising:a) about 0.05 to about 0.5% by weight of human plasmafibronectin, based on the total weight of the gel formulation; and b) awater soluble, pharmaceutically acceptable polymer having a viscosity ofabout 50,000 to about 1,000,000 cps at room temperature.
 2. The aqueousgel formulation of claim 1, wherein the human plasma fibronectin isobtained from autologous, homologous human blood or by recombinant-DNAtechnology.
 3. The aqueous gel formulation of claim 1, having about 0.2to about 0.4% by weight human plasma fibronectin.
 4. The aqueous gelformulation of claim 11, wherein the polymer is selected from the groupconsisting of a vinyl polymer, a polyoxyethylene-polyoxypropylene blockcopolymer, and a cellulose derivative.
 5. The aqueous gel formulation ofclaim 4, wherein the vinyl polymer is a polyacrylic acid.
 6. The aqueousgel formulation of claim 5, wherein the polyacrylic acid has a molecularweight of about 740,000 to about 5,000,000 and is present in an amountin the range from about 0.25 to about 1.0% by weight, based on the totalweight of the gel formulation.
 7. The aqueous gel formulation of claim4, wherein the polyoxyethylene-polyoxypropylene block copolymer has amolecular weight of about 2,000 to about 13,000 and is present in anamount in the range from about 18 to about 35% by weight, based on thetotal weight of the gel formulation.
 8. The aqueous gel formulation ofclaim 4, wherein the cellulose derivative is hydroxypropylcellulose(HPC).
 9. The aqueous gel formulation of claim 8, wherein thehydroxypropylcellulose has a molecular weight of about 370,000 to about1,150,000 and is present in an amount in the range from about 1 to about5% by weight, based on the total weight of the gel formulation.
 10. Theaqueous gel formulation of claim 1, which further comprises apreservative selected from the group consisting of chlorocresol andparabens.
 11. The aqueous gel formulation of claim 10, wherein thechlorocresol is present in an amount in the range from about 0.1 toabout 0.2% by weight and the parabens are present in an amount in therange from about 0.01 to about 0.3% by weight, based on the total weightof the gel formulation.
 12. The aqueous gel formulation of claim 4,which optionally comprises at least one additional ingredient selectedfrom the group consisting of stabilizers and protease inhibitors. 13.The aqueous gel formulation of claim 12, wherein the stabilizer isalbumin, a disaccharide, a cyclic oligosaccharide, or a combinationthereof.
 14. The aqueous gel formulation of claim 13, wherein thealbumin is present in an amount in the range from about 0.1 to about0.1% by weight, the disaccharide is sucrose which is present in anamount in the range from about 0.5 to about 5.0% by weight, and thecyclic oligosaccharide is hydroxypropyl-β-cyclodextrin which is presentin an amount in the range from about 1.0 to about 10.0% by weight, allpercentages by weight being based on the total weight of the gelformulation.
 15. The aqueous gel of claim 12, wherein the proteaseinhibitor is a serine protease inhibitor, a metalloprotease inhibitor,or a combination thereof.
 16. The aqueous gel formulation of claim 15,wherein the serine protease inhibitor comprises about 1.5 to about 45inh U % by weight aprotinin or the metalloprotease inhibitor comprisesabout 0.01 to about 1% by weight EDTA, all percentages by weight beingbased on the total weight of the gel formulation.
 17. A creamformulation for healing chronic wounds, comprising:a) about 0.05 toabout 0.5% by weight of human plasma fibronectin, based on the totalweight of the cream formulation; and b) a cream base having a viscosityof about 60,000 to about 80,000 cps at room temperature.
 18. The creamformulation of claim 17, wherein the cream base has an Abs value ofabout 9.90 and comprises ceteth-20, cetostearyl alcohol, chlorocresol,mineral oil, monobasic sodium phosphate, phosphoric acid, sodiumhydroxide, water, and white petrolatum.
 19. The cream formulation ofclaim 17, wherein the human plasma fibronectin is obtained fromautologous, homologous human blood or by recombinant-DNA technology. 20.The cream formulation of claim 17, having about 0.2 to about 0.4% byweight human plasma fibronectin.
 21. The cream formulation of claim 17,which further comprises a preservative which is chlorocresol which ispresent in an amount in the range from about 0.1 to about 0.2%, based onthe total weight of the cream formulation.
 22. The cream formulation ofclaim 17, which optionally comprises at least one additional ingredientselected from the group consisting of stabilizers and proteaseinhibitors.
 23. The cream formulation of claim 22, wherein thestabilizer is albumin, a disaccharide, a cyclic oligosaccharide, or acombination thereof.
 24. The cream formulation of claim 23, wherein thealbumin is present in the range from about 0.01 to 0.1% by weight, thedisaccharide is sucrose which is present in an amount in the range fromabout 0.5 to about 5.0% by weight, and the cyclic oligosaccharide ishydroxypropyl-β-cyclodextrin which is present in an amount in the rangefrom about 1.0 to about 10.0% by weight, all percentages by weight beingbased on the total weight of the cream formulation.
 25. The creamformulation of claim 22, wherein the protease inhibitor is a serineprotease inhibitor, a metalloprotease inhibitor, or a combinationthereof.
 26. The cream formulation of claim 25, wherein the serineprotease inhibitor comprises about 1.5 to about 45 inh U % by weightaprotinin and the metalloprotease inhibitor comprises about 0.01 toabout 1% by weight EDTA, all percentages by weight being based on thetotal weight of the cream formulation.
 27. An aqueous gel formulationfor topical wound healing, comprising:a) an effective wound healingamount of a wound healing promoter which comprises 0.05-0.5 percent byweight human plasma fibronectin, based on the total weight of the gelformulation; b) a water soluble, pharmaceutically acceptable carrierhaving a viscosity within the range of about 50,000 to about 1,000,000cps at room temperature, wherein the formulation has an Abs valuegreater than 7.8, the Abs value being the percentage of labelled woundhealing promoter present in a deepithelialized dermis sample after 12hours in a diffusion cell system.
 28. An aqueous gel formulation fortopical wound healing, comprising:a) about 0.05 to about 0.5% by weightof a wound healing promotor, based on the total weight of the gelformulation, said wound healing promoter being human plasma fibronectin;b) a water soluble, pharmaceutically acceptable carrier having aviscosity within the range of about 50,000 to about 1,000,000 cps atroom temperature, wherein the formulation has an Abs value of at leastabout 13.4, the Abs value being the percentage of labelled wound healingpromotor present in a deepithelialized dermis sample after 12 hours in adiffusion cell system.
 29. The formulation for topical wound healing ofclaim 27, further comprising an effective amount of a preservative. 30.The formulation for topical wound healing of claim 27, wherein theformulation additionally comprises an extracellular matrix protein. 31.The formulation for topical wound healing of claim 30, wherein theextracellular matrix protein is thrombospondin, laminin, vitronectin, orfibrinogen.
 32. The formulation for topical wound healing of claim 27,wherein the formulation additionally comprises at least one growthfactor.
 33. A method of treating a chronic wound comprising applying theformulation of claim 1 to the wound two times a day.
 34. A method oftreating a chronic wound comprising applying the formulation of claim 17to the wound two times a day.
 35. A method of treating a topical woundcomprising applying the formulation of claim 27 to the wound two times aday.
 36. An aqueous gel formulation for healing chronic wounds,comprising:a) 0.05 to 0.5% by weight of human plasma fibronectin; b)0.25 to 1.0% by weight of polyacrylic acid having a molecular weight ofabout 740,000 to about 5,000,000 and a viscosity of about 350,000 cps,said viscosity being measured at room temperature, and said weightpercentages being based on the total weight of the gel formulation. 37.An aqueous gel formulation for healing chronic wounds comprisingconsisting essentially of 0.2% fibronectin, 0.375% carbomer, and 0.1%chlorocresol, all percentages by weight being based on the total weightof the gel formulation.